作者renshiue (UW-Madison)
看板Biotech
标题Re: [求救] 诱导表现蛋白质
时间Fri May 11 07:31:32 2007
※ 引述《content71 (罗莉饲养中...)》之铭言：
: E. coli strain : BL21(DE3)
: BL21(DE3)pLysS
: Rosetta(DE3)
: induction : E. coli OD600 ~0.4
: 0.5mM, 1mM, for 37度C, 3hr
: result --> BL21(DE3) 在induction後就不太生长，SDS-PAGE check无表现
: BL21(DE3)pLysS 在induction後会继续长，但SDS-PAGE check无表现
: western check在uninduce及induce都有表现但很少,leakage?
: Rosetta(DE3) transformation後就没出现colonies了 (试过两次)
: 我自己的推测是
: 此protein对E.coli有强毒性，所以一但induction菌就会挂掉或不生长
这个会部份表现在DT上。你没测就说没测就好了。
: 现在想到的是 1. 降温induction延长时间
: 2. 换Rosetta(DE3)pLysS (但我们老师肯定不会买...太了解他orz)
: 3. 换表现菌种
: 4. 大量培养，硬是纯化 (最後的办法，算过大概需3 miligram)
蛋白多大？含多少positive charged residues？
另外如果你曾有染到（assuming CBB R-250）过这个蛋白，那麽SDS-PAGE这样的
产量也许还是可以看到, or over-destain?
另外medium也有很大的差别。如果你在LB都养不出来，那我觉得你先去请人带你
作一回比较快。
Transform以前不论的话：
Transform, did you use SOC for 30-60 min recovery?
Your competent cells are cultured by yourself or bought?
inoculation, Did you use single colony or multiple colonies?
Antibiotics storage condition?
(Minimal media) Adding vitamine complex and/or AA complex
(minimal media) How much Glc added?
what's the starting OD? (20mL, or 50mL into 1L) what's the final?
why did you use 0.4? You tested? paper? or former member?
Try small volume first to get the optimal condition or ask him/her.
Try auto-induction media
Try Celtone media etc
SDS-PAGE: boiled for sufficient time? reduced? different staining method?
for purified protein, how does the control/ STD look like?
...too many check points...
personal question: what's the last name of your professor? I may
recommend someone to help you.
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※ 编辑: renshiue 来自: 144.92.4.115 (05/11 07:40)